Apparatus for the treatment of blood by extracorporeal circulation and process of manufacture

ABSTRACT

An apparatus for the treatment of blood or plasma by extracorporeal circulation includes a compartment for the circulation of blood. The compartment is provided with two accesses and has at least one inner surface intended to be coated, after sterilization, with at least one molecular layer of a substance soluble in an aqueous solution. The aqueous solution is capable of increasing the bicompatibility of the inner surface of the compartment. A determined quantity of the substance is deposited inside the blood compartment, at one of the accesses, in a form such that the substance is capable of undergoing, substantially without deterioration, an irradiation capable of sterilizing the apparatus. The quantity of substance is determined such that the circulation of a determined volume of aqueous solution through the blood compartment, starting from the access where the substance has been deposited, should result in the formation, by durable bonding, of at least one molecular layer of the substance on the inner surface of the blood compartment to be treated.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus for the treatment of bloodby extracorporeal circulation and to a process of manufacture of thisapparatus.

2. Description of the Related Art

Apparatuses for the treatment of blood by extracorporeal circulation areemployed in various medical or paramedical applications such as:treatment of renal insufficiency by dialysis or haemofiltration,plasmapheresis and aphaeresis with therapeutic and nontherapeutic aim,blood oxygenation, immunopurification, and the like.

A common feature of all these apparatuses is that they comprise a bloodcompartment provided with two accesses, in which, during the treatmentin question the patient's blood is circulated. To do this, a bloodwithdrawal line is connected between a blood vessel of the patient andan access, employed as entry, of the blood compartment; a blood returnline is connected between the other access of the blood compartment,employed as exit, and a blood vessel of the patient; and the patient'sblood is circulated in this extracorporeal circuit looped onto thepatient, by means of a pump, usually placed in the withdrawal line.

The blood compartment of these apparatuses is generally bounded by aportion of the walls of a casing of the apparatus and by a wall of anactive member of the apparatus, by means of which the treatment of bloodis performed. By way of example, in a dialyser containing hollow fibres,the blood compartment is bounded by the interior of the fibres of abundle of hollow fibres, constituting a semipermeable membrane, by theexternal surface of the discs of adhesive employed for attaching thebundle of fibres to the two ends of a tubular casing of the apparatus,and by two end fittings secured to each end of the casing.

All the materials employed in the manufacture of these apparatuses arechosen to be as biocompatible as possible, so that the reactions(coagulation in particular) which take place when the blood comes intocontact with a foreign material do not take place or take place atrelatively benign levels.

It is known to treat, in bulk or at the surface, the materials intendedto be in contact with blood in order to improve their biocompatibility.The known treatments take place either during the manufacture of somepart or other of an apparatus (bulk treatment), or after the variousparts of the apparatus have been assembled and before sterilization ofthe apparatus, or, extemporaneously, just before the apparatus isemployed.

A problem which is particularly tough to solve arises when attempts aremade to improve the biocompatibility of the active member of anapparatus (for example a dialysis membrane) while conforming to thefollowing conditions:

1) the choice of the substance employed for the treatment and thetreatment methods must result in the modification of a known activemember, this modification having the effect of improving thebiocompatibility of the active member while preserving all the knownqualities (for example, in the case of a dialysis/haemofiltrationmembrane: diffusive and convective transfer performance, adsorptioncapacity for undesirable substances, and the like);

2) sterilization of the apparatus must not affect the treatment;

3) the treatment must not require any special handling by the user.

SUMMARY OF THE INVENTION

The objective of the invention is to propose a process for themanufacture of an apparatus which satisfies these conditions.

More specifically, the objective of the invention is to propose aprocess of manufacture of an apparatus satisfying the conditionsspecified above and in which the active member, before treatment, hasnegative charges at the surface. When blood comes into contact with anegatively charged surface, it is the site of a biological phenomenoncalled activation of the contact phase, which manifests itself in thegeneration of active substances, kallikrein and factor XIIa, frominactive substances, prekallikrein and factor XII.

The activation of the contact phase is benign in itself, but when ittakes place simultaneously with some perturbing factors (taking ofhypotensive medications of IEC type by the patient, dilution of theblood entering the apparatus filled with saline solution, accompanyinglowering of the pH) it seems to be the source of undesirable so-calledanaphylactoid reactions which manifest themselves a few minutes afterthe beginning of the treatment as various symptoms, including an overallfeeling of hotness, numbness of the fingers, the lips or the tongue,panting, nausea and laryngeal oedema. It should be remembered thatanaphylactoid reactions are not exclusively linked with the use ofmedical apparatuses in which the blood compartment has a negativelycharged inner surface. These reactions have been noted with exchangerswhich have membranes of different chemical compositions, sometimes whenfirst employed, sometimes after several utilizations when theexchangers, instead of being discarded after a single use, are usedagain many times and are recycled after each use. An example of anexchanger in which a first use has been accompanied by an undesirablereaction is a dialyser which has a polymethyl methacrylate andpolyacrylonitrile membrane. Reactions associated with the reuse ofdialysers with a cellulose acetate and polysulphone membrane have beenjust as well documented (see "Anaphylactoid reactions associated withreuse of hollow-fiber hemodialyzers and ACE inhibitors" in KidneyInternational, vol. 42 (1992), pp. 1232-1237).

There is provided, in accordance with one aspect of the invention, aprocess for improving the biocompatibility of an apparatus for thetreatment of blood or plasma by extracorporeal circulation. Theapparatus has a compartment for the circulation of blood provided withtwo accesses. In the process, a determined quantity of a substance isdeposited in the blood compartment at at least one of the accesses. Thesubstance is deposited in a form such that the substance is capable ofundergoing, substantially without deterioration, an irradiation capableof sterilizing the apparatus.

The substance is capable of dissolving in an aqueous solution and ofbeing durably bonded to an inner surface of the blood compartment inorder to increase the bicompatibility of the apparatus. The quantity ofsubstance is determined such that the circulation of a determined volumeof aqueous solution through the blood compartment, starting from theaccess where the substance has been deposited, should result in theformation of at least one molecular layer of the substance, by durablebonding, to the inner surface of the blood compartment.

The process also includes sterilizing the apparatus and, before theutilization of the apparatus, circulating a determined volume of aqueoussolution through the blood compartment. The circulation of aqueoussolution starts from the access where the substance has been deposited,so as to carry the substance into contact with the inner surface of theblood compartment and to cause the formation of at least one durablemolecular layer of the substance on the inner surface of the bloodcompartment.

Within the meaning of the invention, the ability to dissolve in anaqueous solution, which is a characteristic of the substance, must beunderstood as a function of the objective to be met. That is at theoutcome of the process, at least one molecular layer of the substance isbonded to the inner surface to be treated of the blood compartment. Inaddition, when it would be undesirable that the substance should beintroduced into the patient's blood, it is necessary that at the outcomeof the process all the substance should have been dissolved and that noresidue should remain in the blood compartment. In other words, thisability comprises both the physical ability to dissolve in an aqueoussolution and a speed of dissolution, which depends on a number ofparameters: volume of aqueous solution employed, temperature of theaqueous solution, and flow rate of the aqueous solution in the bloodcompartment. If these parameters are fixed, for example by deciding thatthe last stage of the process will be carried out immediately before theuse of the apparatus during the procedure for starting up ("priming")the apparatus, then the substance must be chosen, among other criteria,so that the objective of the invention can be met by circulating throughthe blood compartment, for example, two liters of physiological salineat ambient temperature (20° C. to 24° C.) at a flow rate of 200 ml/min(conventional conditions for starting up a dialyser in a dialysiscentre).

The process has two major advantages: on the one hand, the surfacemodified by the substance is obtained only after sterilization of theapparatus, with the result that this modified, biocompatible surfacedoes not run the risk of being damaged by a highly energeticsterilization like sterilization using γ irradiation; on the other hand,when the aqueous solution employed is the solution for starting up theexchanger, the utilization of the apparatus by the user is exactlyidentical with that of any apparatus of the same type.

According to an alternative form of the invention, in an apparatus inwhich the blood compartment has two symmetrical accesses, the determinedquantity of substance is deposited at each access so that, whatever thedirection of subsequent circulation of the aqueous solution through theblood compartment, a molecular layer of the substance is formed on theinner surface to be treated.

Another subject of the invention is a process of manufacture of anapparatus for the treatment of blood or plasma by extracorporealcirculation. The apparatus has a compartment for the circulation ofblood provided with two accesses. The process includes, before a stageof sterilization of the apparatus, the step of depositing in the bloodcompartment, at at least one of the accesses, a determined quantity of asubstance in a form such that the substance is capable of undergoing,substantially without deterioration, an irradiation capable ofsterilizing the apparatus.

The substance is capable of dissolving in an aqueous solution and ofdurably bonding to an inner surface of the blood compartment in order toincrease the biocompatibility of the apparatus. The quantity ofsubstance is determined such that the circulation of a determined volumeof aqueous solution through the blood compartment, starting from theaccess where the substance has been deposited, should result in theformation, by durable bonding, of at least one molecular layer of thesubstance on the inner surface of the blood compartment.

This process has the advantage of being very easy to apply industrially.

Another subject of the invention, is an apparatus for the treatment ofblood or of plasma resulting from the use of the process of manufacturedescribed above.

According to an alternative form of the invention, the surface to betreated has negative charges and the substance is cationic, with theresult that a molecular layer of the surface masks the negative chargesof the surface.

In one embodiment of the invention the apparatus is ahaemodialyser/haemofilter provided with a membrane manufactured from acopolymer of acrylonitrile and of sodium methallylsulphonate (membraneknown under the trade name AN69). The substance chosen for improving thebiocompatibility of this membrane is polyethyleneimine (PEI) which has amolecular mass (measured by light scattering) of between approximately10 and approximately 2000 k dalton. The quantity of PEI (molecular mass25 k dalton) deposited at one or each blood access is preferably betweenapproximately 5 and 10 mg per m² of membrane intended to be in contactwith the blood.

This apparatus does not activate the contact phase and has the sameperformance as an unmodified haemodialyser/haemofilter.

BRIEF DESCRIPTION OF THE DRAWINGS

Other characteristics and advantages of the invention will appear onreading the description which follows. Reference will be made to theattached drawings in which:

FIG. 1 shows a diagrammatic view in lengthwise section of a dialyserwith hollow fibres according to the invention;

FIG. 2 shows the effect of the quantity of PEI employed for treating theAN69 membrane on the electrical surface potential of this membrane;

FIG. 3 shows the effect of the quantity of PEI (molecular mass: 25 kdalton) employed for treating the AN69 membrane on the activation of thecontact phase;

FIG. 4 shows the effect of the quantity of PEI (molecular mass: 750 kdalton) employed for treating the AN69 membrane on the activation of thecontact phase;

FIG. 5 shows the adsorption kinetics of Cytochrome C on a conventionalAN69 membrane and on an AN69 membrane to which PEI has been bonded;

FIG. 6 shows the level of activation of the contact phase by anexperimental membrane devoid of PEI and by the same membrane to whichPEI has been bonded;

FIG. 7 shows the level of activation of the contact phase by a membranebased on polyacrylonitrile devoid of PEI and by the same membrane towhich PEI has been bonded;

FIG. 8 shows the degree of activation of the contact phase by a membranemade of AN69 devoid of DEAE dextran and by the same membrane to whichDEAE dextran has been bonded; and

FIGS. 9a and 9b illustrate the haemocompatibility of a dialysis membraneaccording to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

To illustrate the invention, a particular type of apparatus for theextracorporeal treatment of blood will be described, which is employedfor alleviating renal insufficiency.

A haemodialyser/haemofilter conventionally includes two compartmentsseparated by a semipermeable membrane. A first compartment is intendedto be connected by means of a withdrawal line and a return line to thepatient's vascular circuit, whereas the second compartment has an entryoptionally connected to a source of dialysis liquid (treatment byhaemodialysis and haemodiafiltration) and an exit connected to adischarge for spent liquid (spent dialysate and/or ultrafiltrate). Themembrane is chosen so as to permit the diffusive and/or convectivetransfers of the metabolic wastes from the blood compartment towards thecompartment for spent liquid. The membrane may be manufactured in theform of a planar membrane or of a bundle of hollow fibres. A dialyserwith a planar membrane includes a strip of planar membrane foldedaccordion-style, an insert plate being introduced into all the foldsopening on the same side. As can be seen in FIG. 1, a dialyser withhollow fibres includes a bundle of hollow fibres 1, which is placed in atubular casing 2. The bundle of hollow fibres 1 is held at its two endsby a disc of adhesive 3, 4. In addition to bonding the fibres together,the purpose of the discs of adhesive 3, 4 is to delimit in the tubularcasing 2 a leaktight compartment to which access is given by two smalltubes 5, 6, perpendicular to the axis of the casing 2. At each end ofthe casing 2 is attached an end fitting 7, 8 including a small axialaccess tube 9, 10. The two small tubes 9, 10 are symmetrical. The bloodcompartment of this apparatus consists of the internal space delimitedbetween each disc of adhesive 3, 4 and the end fitting 8, 9 closing thecorresponding end of the tubular casing 2, and by the interior of thehollow fibres.

In accordance with the invention, to improve the biocompatibility ofthis apparatus by modifying an inner surface of the blood compartment, adetermined quantity of a substance soluble in an aqueous solution andcapable of modifying the surface involved in the desired manner isdeposited in each end fitting 8, 9 after the apparatus has beenassembled as shown in FIG. 1. The quantity of substance is chosen suchthat, after circulation of a determined quantity of aqueous solutionthrough the blood compartment, at least one molecular layer of thesubstance covers the surface whose biocompatibility is to be improved.The quantity of substance is deposited in the form of a drop 11, 12 bymeans of a conventional injection device. The drop is formed either by agel of the substance or by a matrix material in which the substance isincorporated.

After deposition of the substance has been performed, the small accesstubes 5, 6, 9, 10 are stoppered and the apparatus can be sterilized, forexample by means of ethylene oxide or by γ irradiation.

The use of any apparatus for the treatment of blood or plasma byextracorporeal circulation includes a preliminary starting-up stageduring which the blood compartment is rinsed and filled with a sterileaqueous solution.

In accordance with the invention, this stage of preparation of theapparatus is exploited for dissolving the treatment substance andbringing it into contact with the surface to be treated. Before thebeginning of the actual dialysis session, the user connects one of thesmall access tubes 9 (10) of the blood circuit to a pouch of sterilesolution, connects the other small access tube 10 (9) to an emptycollecting pouch, and makes the sterile solution circulate through theblood compartment, if appropriate by means of the blood pump of thedialysis machine. The sterile solution dissolves the substance andcarries it into contact with the surface to be treated, where it isbonded, for example by ionic or covalent bonding.

In the exemplary of embodiment just described, it is because the twosmall tubes 9, 10 for access to the blood compartment are symmetrical soas not to have to impose on the user a direction of circulation of thesaline solution when the haemodialyser is being started up, that aquantity of appropriate substance (drops 11, 12) has been deposited ineach of the end fittings. The substance which is deposited in thedownstream end fitting during the start-up is therefore not employed forthe treatment of the surface whose biocompatibility is to be improved.Naturally, by suitably marking the end fitting to which the pouch ofsaline solution is to be connected for starting up, it is possible todeposit the drop of the treatment substance in only one end fitting 7,8.

According to an alternative form of the invention, instead ofmanufacturing a blood treatment apparatus according to the processdescribed above, where a drop of appropriate substance is deposited inat least one of the small tubes 9, 10 for access to the bloodcompartment, a coupling is manufactured comprising a female portionmatching these small tubes 9, 10, and a male portion matching the femaleconnection member fitted to the end of the lines employed for connectinga patient to a blood treatment apparatus. This coupling is obtained bymoulding or extruding a plastic which is suitable for contact withblood. A drop of the substance intended to improve the biocompatabilityof a blood treatment apparatus is deposited inside this coupling. Thecoupling is next placed in a packaging capable of forming a sterilebarrier and then the whole is sterilized by γ irradiation. The couplingis fitted to a conventional blood treatment apparatus before thepreliminary stage, described above, of starting-up the apparatus.

In one embodiment of the invention the portion of the haemodialyserwhose biocompatibility is to be improved is a semipermeable membranewhose surface has negative charges. The aim of the improvement is toprevent the activation of the contact phase or to neutralize it. Inaccordance with the invention an appropriate substance exhibits thefollowing characteristics:

1--it must be cationic, so as to bond to the membrane by ionic bondingand to mask negative charges on the surface of the membrane;

2--it must be soluble in water so as to be dissolvable in the aqueoussolution employed for starting-up the apparatus;

3--it must not be toxic;

4--it must be macromolecular and its size must be chosen in such a waythat a macromolecule does not enter the pores of the membrane (forexample, in the case of the AN69 membrane, this size must be at least 10k dalton). In the course of utilization, a macromolecule attached to themembrane will be difficult to displace from it with a blood protein.Also, a macromolecule which is prohibited by its size from enteringbiological cells is a priori less toxic than a molecule whose dimensionsallow it to enter a cell;

5--it must, where appropriate, withstand an energetic sterilization, ofthe γ irradiation type, even if it is partially affected thereby. Inother words, at least a proportion of the molecules must remain intactand be capable of bonding to the membrane in the desired manner.Furthermore, the irradiated substance must not become toxic;

6--once bonded to the membrane, it must not significantly alter thecharacteristics of the membrane (haemocompatibility, diffusive andconvective transfer capacity, protein adsorption capacity).

To give an example, it has been discovered, in accordance with theinvention, that PEI with a molecular mass of between 10 and 2000 kdalton, which is in the form of a water-soluble gel, is a whollyappropriate substance for the modification of the membrane known underthe trade name AN69, the surface of which has negative charges. Inparticular, in-vitro tests have shown that the potential toxicitythreshold of irradiated PEI corresponds to the injection of a quantityof PEI needed to raise the PEI concentration of an internal liquid(plasma) to 0.4 mg/ml (by way of comparison, if the quantity of PEIsufficient to treat a conventional dialyser, that is 10 mg, wereinjected into the blood of an adult, the PEI concentration in the plasmawould be of the order of 0.002 mg/ml).

Another appropriate substance for the modification of the membrane knownunder the trade name AN69 is diethylaminoethyl dextran (DEAE dextran)with a mean molecular mass of 500 k dalton. Unlike PEI, this substanceis not available in the form of a water-soluble gel, but of a powder.Thus, it is necessary to incorporate it into a gel of a neutral matrixmaterial in order to be capable of employing it in accordance with theinvention. To give an example, a carboxymethylcellulose gel can beemployed for this purpose.

The main stages of the manufacture of a hollow fibre made of AN69 arerecalled briefly. A solution of polymer is prepared, containing 35% byweight of a copolymer of acrylonitrile and of sodiummethallylsulphonate, 52% by weight of dimethylformamide (DMF) and 13% byweight of glycerol. The polymer solution is heated to 130° C. and isextruded through a die which has two concentric nozzles, nitrogen beinginjected into the inner nozzle to form the aperture of the hollow fibre.Upon contact with the ambient air (approximately 20-25° C.), thethermoreversible gel fibre leaving the die is the site of a thermalphase inversion. The fibre is next received in a water bath in which thesolvent (DMF) in the fibre is replaced with water. The fibre is nextimmersed in hot water at 95° C., where it is drawn approximatelyfourfold. A stage of stabilization in hot water at 95° C. follows.Finally, the fibre is glycerinated with a water/glycerol mixture.

The manufacture of a planar membrane from AN69 comprises the followingstages. A solution of polymer is prepared, containing 21% by weight of acopolymer of acrylonitrile and of sodium methallylsulphonate and 79% byweight of dimethylformamide (DMF). After filtration and degassing, thispolymer solution is extruded by means of a slot-shaped die onto a rotaryroll heated to 80° C. Some of the DMF is evaporated off. The filmobtained is drawn approximately three-and-a-halffold in hot water at 95°C. A stage of stabilization in hot water at 95° C. follows. Finally themembrane is glycerinated in a water/glycerol mixture.

EXAMPLE 1

A dialyser including approximately 8500 hollow AN69 fibres wasassembled. Each fibre had the following dimensions: inner diameter: 240μm, wall thickness: 50 μm, length: 0.225 m. The area of the membraneintended to come into contact with the blood was approximately 1.44 m².A drop of 10 mg of PEI (BASF Lupasol WF, molecular mass: 25 k dalton)was deposited in each small tube for access to the blood compartment,after which the tubes for access to the two compartments of the dialyserwere closed with special stoppers and the dialyser was sterilized withethylene oxide. Two liters of sterile physiological saline (solution ofsodium chloride at a concentration of 0.9 g/l) at ambient temperature(22° C.) were circulated at a flow rate of 200 ml/min through the bloodcompartment of this dialyser. The physiological saline dissolved thedrop of PEI gel and the PEI molecules circulated through the dialyserwere bonded, by ionic bonding, to the sodium methallylsulphonate groupspresent at the surface of the membrane.

To measure whether the dialyser thus manufactured and prepared for usehad the level of biocompatibility set as objective of the invention,this dialyser was subjected to the following test: a biological liquidwas prepared, capable of stimulating the production of kallikreins incontact with a membrane charged negatively at the surface. Thebiological liquid used for the test consisted of human plasma lean inplatelets, diluted to 5% in physiological saline containing addedcitrate (it is noted that the conditions of the test employed are farfrom the conditions of use of an apparatus for extracorporeal bloodcirculation: the dilution ratio is very high, the liquid chosen isplasma and not blood, the plasma contains added citrate and is henceacidified, whereas in dialysis the anticoagulant employed is heparin.These test conditions are chosen deliberately because they stimulate andamplify the activation of the contact phase). One and a half liters ofthis liquid were circulated in a closed circuit through the bloodcompartment of the dialyser at a flow rate of 100 ml/min for six hours.The plasma kallikreins were assayed in samples of liquid takenintermittently by means of a conventional chromogenic test, startingwith the S 2302 substrate from the Biogenic company. The result of thedeterminations shows unequivocally that the dialyser manufactured inaccordance with the invention does not cause a rise in the content ofkallikreins in a dilute plasma.

It is pointed out that, bearing in mind the sensitivity of thechromogenic test employed, it is considered that there is no significantrise in the content of kallikreins if the kallikrein concentrationremains below approximately 10 units per liter.

It is emphasized that, besides its efficiency insofar as the improvementin the biocompatibility of the dialyser described above is concerned,this process of manufacture has a major advantage, even whensterilization with ethylene oxide would in principle make it possible toattach the PEI to the membrane before the sterilization stage. In fact,bearing in mind that the AN69 fibres are glycerinated, if they were tobe treated with PEI before the sterilization of the dialyser, it wouldbe necessary:

1--to deglycerinate the fibres by rinsing the dialyser with an aqueoussolution;

2--to circulate a solution of PEI through the blood compartment;

3--to reglycerinate the fibres in order to expel water therefrom, sinceethylene oxide has no effect on a wet product; and

4--to purge the fibres of the excess glycerol.

Apart from the fact that the fourth stage would be difficult or evenimpossible to carry out, it will be understood that adding fouradditional stages to an industrial manufacturing process would make itscost prohibitive. To summarize, the process according to the invention,according to which the deglycerinating of the fibres and their treatmentwith PEI are done simultaneously when the dialyser is being started up,makes it possible to industrialize the improvement in thebiocompatibility of a dialyser whose membrane is made of AN69.

EXAMPLE 2

A dialyser with a planar AN69 membrane was assembled. The membrane had athickness of approximately 20 μm. The area of the membrane intended tocome into contact with the blood was approximately 1.50 m². A drop of 10mg of PEI (Lupasol WF, 25 k dalton) was deposited in each small tube foraccess to the blood compartment, after which the small tubes for accessto the two compartments of the dialyser were closed off and the dialyserwas sterilized by γ irradiation (36 kGy). Two liters of sterilephysiological saline at ambient temperature (approximately 22° C.) werecirculated through the blood compartment of this dialyser. Thephysiological saline dissolved the drop of PEI gel and the PEI moleculescirculated through the dialyser were bonded to the sodiummethallylsulphonate groups present at the surface of the membrane.

The dialyser was then subjected to the in-vitro test described inExample 1, intended to measure whether the AN69 membrane coated with amonolayer of PEI activates the contact phase. As in the case of thedialyser with hollow fibres, the test result was negative.

EXAMPLE 3

The graph in FIG. 2 shows the result of in-vitro tests performed on adialyser with a planar membrane of the type in Example 2, intended todetermine the effect of the quantity of PEI (Lupasol WF, 25 k dalton)used per dialyser on the electrical surface charge of a membrane made ofAN69. The electrical surface charge is evaluated by measuring the flowpotential, as defined below: the starting point is that a solution ofelectrolyte circulating through the compartment of a dialyser generatesa potential difference DE proportional to the pressure drop DP createdby the electrolyte solution between the entry and the exit of thedialyser. After the dialyser has been rinsed and when its membrane hastherefore been coated with PEI, a solution of sodium chloride (10⁻² M)was circulated through the blood compartment and DE and DP were measuredat the access points to the blood compartment, by means of Ag/AgClelectrodes and pressure sensors. The DE/DP ratio is called the flowpotential of the electrolyte in the dialyser and this ratio ischaracteristic of the surface charge of the membrane.

In this diagram it can be seen that approximately 5 mg of PEI aresufficient to make the surface of the membrane electrically neutral.

EXAMPLE 4

The graph in FIG. 4 shows the result of in-vitro tests performed on adialyser with a planar membrane of the type in Example 2, intended todetermine the effect of the quantity of PEI (Lupasol WF, 25 k dalton)used per dialyser on the activation of the contact phase. The activationof the contact phase is evaluated by means of the generation ofkallikreins according to the test described in Example 1. In thisdiagram it is seen that 10 mg of PEI (molecular mass 25 k dalton) aresufficient to completely suppress the activation of the contact phase.

EXAMPLE 5

The graph in FIG. 4 shows the result of in-vitro tests performed on adialyser with a planar membrane of the type in Example 2, intended todetermine the effect of the quantity of PEI (Lupasol P, 750 k dalton)used per dialyser on the activation of the contact phase. The activationof the contact phase is evaluated by means of the generation ofkallikreins according to the test described in Example 1. In thisdiagram it is seen that 7 mg of PEI (molecular mass 750 k dalton) aresufficient to completely suppress the activation of the contact phase.

EXAMPLE 6

By employing cytochrome C as test molecule it was verified in vitro thatthe protein adsorption capacity within the bulk of the membrane remainsunchanged after treatment of the membrane with PEI (Lupasol WF, 25 kdalton) with 10 mg per dialyser with a planar AN69 membrane which has anarea of approximately 1.25 m². To perform this verification, thefollowing test was carried out on two dialysers with a planar AN69membrane (area of approximately 1.25 m²), one of which had been treatedwith PEI and the other had not: six liters of a solution of cytochrome Cat a concentration of 20 mg/l in a pH 7.4 phosphate buffer medium werecirculated, in open circuit, at a flow rate of 300 ml/min, through theblood compartment of each dialyser. The concentration of cytochrome C inthe solution leaving the dialyser was measured at regular time intervalsand the cumulative quantity of cytochrome C adsorbed was calculated.FIG. 5 shows that the result of the measurements was the same for bothdialysers.

EXAMPLE 7

It was also verified in vitro that the bonding of PEI to the membranewas completely irreversible and that PEI was not released back into aliquid circulated through the blood compartment of the dialyser. Toperform this verification, two independent tests were performed:

1 st test: the flow potential of a solution of sodium chloride wasmeasured (according to the method in Example 3) in the blood compartmentof a dialyser of the type described in Example 2. Physiological salinewas next circulated in open circuit at 37° C. at a flow rate of 300ml/min for 5 hours, through the blood compartment of the dialyser. Theflow potential of a sodium chloride solution was then measured again andthe results of the two measurements were compared. Since they wereidentical, it was concluded that the bonding between the AN69 membraneand the PEI is durable.

2nd test: PEI (Lupasol P, 750 k dalton) was labelled with a moleculecontaining a radioactive isotope ([2,3-³ H ]N-succimidyl propionate) byproceeding as follows: PEI was reacted with [2,3-³ H]N-succimidylpropionate to obtain ³ H-PEI (labelled PEI) which was next precipitatedwith sulphosalicylic acid, to separate the labelled PEI from the excess[2,3-³ H]N-succimidyl propionate; the precipitate was then subjected tosuccessive washings with water to remove the excess of [2,3-³H]N-succimidyl propionate and to obtain labelled PEI which had aspecific activity of 1.52 mCi/g (which corresponds to a meansubstitution ratio of 11.7 mmol of 3H propionate per μmol of PEI).

A dialyser including approximately 8500 hollow AN69 fibres wasmanufactured in accordance with the process described in the firstparagraph of Example 1, the only difference being that it was one dropof 10 mg of labelled PEI that was deposited in each small tube foraccess to the blood compartment of the dialyser, instead of standard PEI(BASF Lupasol WF, molecular mass: 25 k dalton).

Through the blood compartment of this dialyser was circulated in closedcircuit for four hours 0.51 of whole human blood containing heparin at aconcentration of 3 IU/ml (standard heparin marketed by PAN Pharma). Asample of blood circulating through the dialyser was taken every halfhour and its radioactivity was measured. No desorption of labelled PEIwas detected in any sample (bearing in mind the means of measurementemployed, it is considered that the detection limit was 0.4 μg/ml).

EXAMPLE 8

An experimental membrane M was manufactured in the form of a hollowfibre according to the following stages. A solution of polymer wasprepared containing 28% by weight of a copolymer of acrylonitrile andvinyl acetate, 51% by weight of dimethylformamide (DMF) and 21% byweight of glycerol. The polymer solution was heated to 120° C. and wasextruded through a die which had two concentric nozzles, nitrogen beinginjected into the inner nozzle to form the aperture of the hollow fibre.Upon contact with the ambient air (approximately 20-25° C.) thethermoreversible gel fibre leaving the die was the site of a thermalphase inversion. The fibre was next received in a water bath in whichthe solvent (DMF) in the fibre was replaced with water. The fibre wasnext immersed in hot water at 40° C., where it was drawn approximatelytwofold. The resulting fibre was next stabilized in hot water at 60° C.Finally the fibre was glycerinated in a water/glycerol mixture.

A dialyser including approximately 10,000 hollow M fibres was assembled.Each fibre had the following dimensions: inner diameter: 190 μm; wallthickness: 50 μm, length: 0.225 m. The area of the membrane intended tocome into contact with the blood was approximately 1.34 m². A drop of 12mg of PEI (Lupasol WF; molecular mass: 25 k dalton) was deposited ineach small tube for access to the blood compartment. Two liters ofsterile physiological saline (solution of sodium chloride at aconcentration of 0.9 g/l) at ambient temperature (22° C.) werecirculated at a flow rate of 200 ml/min through the blood compartment ofthis dialyser. The physiological saline dissolved the drop of PEI geland the PEI molecules circulated through the dialyser were bonded, byionic bonding, to the surface of the membrane.

The dialyser was then subjected to the in-vitro test described inExample 1, intended to measure whether the M membrane coated with a PEImonolayer activates the contact phase. As in the case of the dialyserwith hollow fibres, the test result was negative. FIG. 6 shows theactivation level caused by this dialyser and the activation level causedby a dialyser of the same manufacture but whose membrane has not beencoated with PEI.

EXAMPLE 9

The effectiveness of the invention was verified on a dialyser withhollow fibres, equipped with a commercially availablepolyacrylonitrile-based membrane, namely a PAN 13 DX model dialyser(membrane working area: 1.3 m²) manufactured by Asahi. This dialyser wastreated in accordance with the process described in the first paragraphof Example 1, the only difference being that it was one drop of 10 mg ofPEI of molecular mass 750 k dalton (BASF Lupasol P) that was depositedin the small tube for entry to the blood compartment of the dialyser,instead of PEI of molecular mass 25 k dalton (BASF Lupasol WF).

The PAN 13 DX model dialyser was then subjected to the in-vitro testdescribed in Example 1, intended to measure whether the membrane coatedwith a PEI monolayer activates the contact phase. The test result wasnegative. FIG. 7 shows the activation level caused by this dialyser andthe activation level caused by a dialyser of the same manufacture butwhose membrane has not been coated with PEI.

EXAMPLE 10

The feasibility of the invention was verified by employing, as substanceintended to modify a membrane, not a substance available in gel form(like PEI), but a substance available in powder form.

A water-soluble gel which was nontoxic and chemically neutral towardsthe AN69 membrane was prepared by dissolving carboxymethylcellulose(C5678 from Sigma) at ambient temperature (25° C.) in demineralizedwater in a proportion of 12.5 mass % of carboxymethylcellulose and 87.5mass % of water. Into this neutral matrix was incorporated, as activesubstance, diethylaminoethyl dextran (D 1162 from Sigma) of meanmolecular mass 500 k dalton, in a proportion of 500 mass % of DEAEdextran and 50 mass % of carboxymethylcellulose gel.

A dialyser including approximately 8500 hollow AN69 fibres wasassembled. Each fibre had the following dimensions: inner diameter: 240μm; wall thickness: 50 μm; length: 0.225 m. The area of the membraneintended to come into contact with the blood was approximately 1.44 m².A drop of 100 mg of the neutral gel/active substance mixture (that is 50mg of DEAE dextran) was deposited in a small tube for access to theblood compartment. Two liters of sterile physiological saline (solutionof sodium chloride at a concentration of 0.9 g/l) at ambient temperature(25° C.) were circulated at a flow rate of 200 ml/min through the bloodcompartment of this dialyser starting from the access where the drop ofneutral gel/active substance mixture was deposited. The physiologicalsaline dissolved the drop of gel and the DEAE dextran moleculescirculated through the dialyser were bonded, by ionic bonding, to thesodium methallylsulphonate groups present at the surface of themembrane.

To measure whether and--where appropriate--to what extent, the dialyserthus manufactured activated the contact phase, it was subjected to thetest described in Example 1, the only variant being that the humanplasma diluted to 5% in physiological saline was circulated through theblood compartment of the dialyser for one hour instead of six, which wassufficient, bearing in mind the objective of the verification performed.The result of the determinations shows unequivocally that this dialyserproduces only a weak activation of the contact phase (FIG. 8). It shouldbe noted that, bearing in mind the objective aimed at, no attempt wasmade in this example to optimize the parameters which make it possibleto obtain the total neutralization of the contact phase, in particularthe quantity of active substance and the viscosity of the matrix gel.

To finish with, the dialyser treated with PEI was subjected to a wholeseries of conventional measurements intended to determine itscharacteristics: toxicity, haemocompatibility, diffusive and convectivetransfer capacity and the like. The characteristics of the treateddialyser were at least as good as those of an untreated dialyser of thesame type.

Toxicity test

Two batches of dialysers which had a membrane made up of hollow AN69fibres were assembled, each fibre having the following dimensions: innerdiameter: 210 μm, wall thickness: 42 μm. The dialysers of the firstbatch included approximately 9024 fibres (length: 0.24 m) and thedialysers of the second batch included approximately 11520 fibres(length: 0.30 m), the area of the membrane intended to come into contactwith the blood being approximately 1.43 m² in the first case andapproximately 2.26 m² in the second case. Each dialyser was nextsubjected to a treatment including the stages of:

1) deglycerinating the fibres by rinsing the dialyser with water;

2) circulating for 6 min through the blood compartment, in open circuit,at a flow rate of 200 ml/min, an aqueous solution of PEI (Lupasol WF,molecular mass: 25 k dalton) at a concentration of 1 g/l. At the end ofthis stage approximately 340 mg of PEI were attached to the membrane ofa dialyser of the first batch (approximately 9024 fibres) andapproximately 500 mg of PEI were attached to the membrane of a dialyserof the second batch (approximately 11520 fibres);

3) sterilizing the dialyser by γ irradiation (36 kGy).

It is noted that the quantity of PEI attached to the membrane of thesedialysers was much greater than the quantity attached to the membrane ofthe dialysers of Examples 1, 2 and 8 and that the sterilization wasperformed when the PEI was already bonded to the membrane.

These dialysers satisfied the tests for biological evaluation of medicaldevices, defined in international standard ISO 10993-1.

Evaluation of haemocompatibility

The haemocompatibility of a dialysis membrane, and in particular itsnonthrombogenic nature is linked with its protein adsorption propertiesat the surface of the membrane in contact with the blood.

The adsorption kinetics of fibrinogen labelled with iodine 125 weremeasured in vitro on small-scale dialysers which had a membrane made upof AN69 fibres, in hydrodynamic conditions comparable with those of adialysis session.

The small-scale model dialysers employed for the test included 170 AN69fibres (inner diameter: 210 μm, wall thickness: 42 μm, length: 0.18 m)whose surface intended to come into contact with the blood had beentreated with PEI in accordance with the invention (approximately 30mg/m², respectively 300 mg/m², in the case of the minidialysers in theblood compartment of which the drop of PEI deposited was of 0.7 mg,respectively of 7 mg).

Heparin-containing human blood containing radiolabelled fibrinogen in aconcentration of 2.5 μg/ml was prepared and this liquid was circulated,in open circuit, at a flow rate of 2.5 ml/min in a control minidialyser(AN69 without PEI) and in a minidialyser according to the invention. Therate of absorption of the fibrinogen, determined by means of themeasurement of the radioactivity of the minidialyser, is shown in FIG.8b. It was found that this rate was substantially the same for a controlminidialyser and for the minidialysers to the membrane of which PEI hasbeen attached.

The same test was performed with a solution of radioactively labelledfibrinogen in a pH 7.4 buffer liquid (concentration of 2.5 μg/ml). Ascan be seen in FIG. 8a, here, too, the rate of adsorption issubstantially the same for a control minidialyser and for a minidialyserto the membrane of which PEI has been attached.

Convective transfer capacity

The ultrafiltration gradient and the maximum ultrafiltration flow ratewere measured for two control dialysers (No. 1 and 2) which had a planarAN69 membrane with an area of 1.53 m² and two dialysers (No. 3 and 4)which had a planar AN69 membrane with an area of 1.53 m², preparedaccording to the process described in Example 2 with 12 mg of PEI (BASFLupasol WF, molecular mass: 25 k dalton).

The measurement of the ultrafiltration gradient and the measurement ofthe maximum ultrafiltration flow rate were performed in the followingmanner: heparin-containing and standardized ox blood (protein content of60 g/l and hematocrit of 32%) was circulated through the bloodcompartment of the dialyser at the steady flow rate of 300 ml/min. Anultrafiltration of the blood through the membrane was produced by meansof a pump connected to the second compartment of the dialyser. Bygradually increasing the ultrafiltration flow rate the transmembranepressure (TMP) is increased and is measured continuously with the aid oftwo pressure sensors connected respectively to the two compartments ofthe dialyser, and the ultrafiltration gradient in ml/h mm Hg is deducedfrom this. Starting from a threshold value, the ultrafiltration flowrate remains stable even if the TMP continues to increase. The maximumultrafiltration flow rate in ml/min is then measured.

The table below shows the result of these measurements, which shows thatthe conventional dialysers and the dialysers according to the inventionhave equivalent convective transfer capacities.

    ______________________________________                                                    Ultrafiltration                                                                          Maximum ultra-                                         Dialyser    gradient   filtration flow                                        No.         (ml/h mm Hg                                                                              rate (ml/min)                                          ______________________________________                                        1           39         100                                                    2           43         104                                                    3           43         107                                                    4           45         104                                                    ______________________________________                                    

The present invention is not limited to the example just described andcan have alternative forms.

We claim:
 1. Apparatus for the treatment of blood or plasma byextracorporeal circulation, comprising:a compartment for the circulationof blood, provided with two accesses, the compartment having at leastone inner surface intended to be coated, after sterilization, with atleast one molecular layer of a substance soluble in an aqueous solutionand capable of increasing biocompatibility of the at least one innersurface; and a determined quantity of the substance deposited inside theblood compartment, at one of the accesses, in a form such that thesubstance is capable of undergoing, substantially without deterioration,an irradiation capable of sterilizing the apparatus, the determinedquantity of the substance being determined such that the circulation ofa determined volume of aqueous solution through the blood compartment,starting from the access where the determined quantity of the substancehas been deposited, should result in the formation, by durable bonding,of at least one molecular layer of the substance on the at least oneinner surface of the blood compartment.
 2. Apparatus according to claim1, wherein the two accesses are symmetrical, and wherein the determinedquantity of the substance is deposited at each access so that, whateverthe direction of the subsequent circulation of the aqueous solutionthrough the blood compartment, the molecular layer of the substance isformed on the inner surface to be treated.
 3. Apparatus according toclaim 1, wherein the determined quantity of the substance is in the formof a drop of a gel of the substance.
 4. Apparatus according to claim 1,wherein the determined quantity of the substance is in the form of adrop of a matrix material in which the substance is incorporated. 5.Apparatus according to claim 1, wherein the inner surface to be treatedhas negative charges, and wherein the substance is cationic, with theresult that a molecular layer of the substance masks the negativecharges of the surface.
 6. Apparatus according to claim 1, wherein theapparatus include a haemodialyser/haemofilter including two compartmentsseparated by a semipermeable membrane, a first compartment being thecompartment for the circulation of bloods and a second compartment beingintended for the circulation of spent liquid, wherein the inner surfaceto be treated is the surface of the semipermeable membrane situatedwithin the first compartment.
 7. Apparatus according to claim 6, whereinthe membrane is manufactured from an acrylonitrile copolymer, andwherein the substance is polyethyleneimine.
 8. Apparatus according toclaim 7, wherein the polyethyleneimine has a molecular mass of betweenapproximately 10 and approximately 2000 k dalton.
 9. Apparatus accordingto claim 8, wherein the polyethyleneimine has a molecular mass of 25 kdalton.
 10. Apparatus according to claim 8, wherein thepolyethyleneimine has a molecular mass of 750 k dalton.
 11. Apparatusaccording to claim 8, wherein the quantity of polyethyleneimine ofmolecular mass of 25 k dalton deposited at one or each of the accessesis between approximately 5 mg and approximately 10 mg per m² of activemembrane.
 12. Apparatus according to claim 6, wherein the membrane is aplanar membrane.
 13. Apparatus according to claim 6, wherein themembrane consists of a bundle of hollow fibres.
 14. Process ofmanufacture of an apparatus for the treatment of blood or plasma byextracorporeal circulation, the apparatus having a compartment for thecirculation of blood provided with two accesses, the processcomprising:before a step of sterilization of the apparatus, the step ofdepositing in the blood compartment, at at least one of the accesses, adetermined quantity of a substance in a form such that the substance iscapable of undergoing, substantially without deterioration, anirradiation capable of sterilizing the apparatus, the substance beingcapable of dissolving in an aqueous solution and of durably bonding toan inner surface of the blood compartment in order to increasebicompatibility of said inner surface, the determined quantity of thesubstance being determined such that the circulation of a determinedvolume of aqueous solution through the blood compartment, starting fromthe access where the determined quantity of the substance has beendeposited, should result in the formation, by durable bonding, of atleast one molecular layer of the substance on said inner surface of theblood compartment.
 15. Process for improving the biocompatibility of anapparatus for the treatment of blood or plasma by extracorporealcirculation, the apparatus having a compartment for the circulation ofblood provided with two accesses, the process comprising the stepsof:depositing in the blood compartment, at at least one of the accesses,a determined quantity of a substance in a form such that the substanceis capable of undergoing, substantially without deterioration, anirradiation capable of sterilizing the apparatus, the substance beingcapable of dissolving in an aqueous solution and of durably bonding toan inner surface of the blood compartment in order to increasebiocompatibility of the inner surface of the blood compartment, thedetermined quantity of the substance being determined such that thecirculation of a determined volume of aqueous solution through the bloodcompartment, starting from the access where the determined quantity ofthe substance has been deposited, should result in the formation, bydurable bonding, of at least one molecular layer of the substance on theinner surface of the blood compartment; sterilizing the apparatus; andbefore the utilization of the apparatus, circulating the determinedvolume of aqueous solution through the blood compartment, starting fromthe access where the determined quantity of the substance has beendeposited, so as to carry the substance into contact with the innersurface of the blood compartment and to cause the formation of at leastone durable molecular layer of the substance on the inner surface of theblood compartment.
 16. Process according to either of claim 14 or claim15, wherein the two accesses are symmetrical, and wherein the determinedquantity of the substance is deposited at each access so that, whateverthe direction of the subsequent circulation of the aqueous solutionthrough the blood compartment, the molecular layer of the substance isformed on the inner surface to be treated.
 17. Process according toeither claim 14 or claim 15, wherein the determined quantity of thesubstance is deposited in the form of a drop of a gel of the substance.18. Process of manufacture according to either claim 14 or claim 15,wherein the determined quantity of the substance is deposited in theform of a drop of a matrix material in which the substance isincorporated, the matrix material being soluble in an aqueous solution.19. Process according to claim 15, wherein the apparatus is sterilizedby means of ethylene oxide.
 20. Process according to claim 15, whereinthe apparatus is sterilized by γ irradiation.